Isolation and Incorporation of the Kv2.2 Gene
into Mouse Potassium Channels
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Anuj Raj Shah |
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Potassium channels play a key role in transmitting the nervous system's electrical signals, which control the hearts pace, hormone balance and several other biological processes. The mouse genes code for several different types of potassium channels. One of these genes is the Kv2.2 gene which codes for a channel that has a slow activation and almost no inactivation.. These genes are a closely related group, and because of their heterogeneous nature, each gene is very similar to the rest and they are immensely varied.
This Kv2.2 potassium channel gene was produce using a TOPO vector and was cut using restriction enzymes. Once Kv2.2 was isolated and placed into a PBK-CMV (Stratagene) Vector, it was introduced into competent Ecoli cells. These cells where then lysed and tested to see if the Kv2.2/PBK-CMV ligation was successful and To see if the insert was in the right orientation. Throughout the experimentation, the tests showed that the insert was always in the wrong orientation, or not inserted at all. This procedure was attempted several times and with several different plasmids, yet each time the insert was either defective or not present. The reason for this is not completely known, but one logical explanation is that the enzyme used to cut the Kv2.2 fragment out was too similar to the enzyme used to cut the plasmid for ligation and therefore the plasmid could not have properly inserted. This problem hindered the experiment and the experiment could not progress beyond this point.
In the future, we expect to use a different restriction enzyme during ligation. This will facilitate the bonding of the Kv2.2 gene to the plasmid much more than before. Once this is achieved, and the plasmid is inserted into the Ecoli competent cells, and the orientation of the insert is shown to be correct, then we should be able to grow up the bacteria and extract the DNA. This DNA may then be used to transform mice. Once the mice have grown up, mated, and produced offspring, then the offspring may be tested for the new Kv2.2 potassium channel traits. These potassium channels will be tested for membrane potential and various other tests. This work will lead us to a better understanding of the heart and how it regulates the electron signals from the brain. If the Kv2.2 gene is found to conduct the hearts rhythm much more effectively than those with other genes do, then this procedure may be able to be adapted to other animals, even humans
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Washington University - Biology
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